利用生物发光成像示踪大鼠颅骨缺损中移植的骨髓间充质干细胞  

Tracing transplanted bone marrow mesenchymal stem cells in rat calvarial defect by bioluminescence imaging

作　　者：曾裕威 黄闯 魏建国 段东明 王簕[1] Zeng Yuwei;Huang Chuang;Wei Jianguo;Duan Dongming;Wang Le(Department of Orthopedics,Third Affiliated Hospital of Guangzhou Medical University,Guangzhou 510150,Guangdong Province,China)

机构地区：[1]广州医科大学附属第三医院骨科,广东省广州市510150

出　　处：《中国组织工程研究》2022年第25期3968-3973,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(82072409),项目负责人:王簕;广东省自然科学基金(2019A1515012020),项目负责人:王簕。

摘　　要：背景:生物发光成像是一种新兴的活体成像技术,目前尚未有运用该技术对移植干细胞在颅骨缺损活体示踪的研究。目的:探究Luciferase慢病毒转染大鼠骨髓间充质干细胞的最佳转染复数,进一步研究表达荧光素酶骨髓间充质干细胞在体外的有效性和稳定性以及在大鼠颅骨缺损内的示踪作用。方法:以不同感染复数转染对数生长期的大鼠骨髓间充质干细胞,并在活体成像系统中定量分析荧光值以确定最佳感染复数;在最佳感染复数下构建表达荧光素酶的骨髓间充质干细胞,分析不同数量细胞和不同代数细胞对应的荧光值变化,以评估转染干细胞的有效性和稳定性;最后在颅骨缺损中示踪表达荧光素酶的骨髓间充质干细胞。结果与结论:①感染复数在10-50区间递增时,荧光定量随感染复数同比例增加,而感染复数增加至100时,荧光定量增幅不明显,证明Luciferase慢病毒转染大鼠骨髓间充质干细胞的最佳感染复数为50;②表达荧光素酶的骨髓间充质干细胞在体外具有良好的细胞-生物荧光量效关系和增殖稳定性,证明荧光定量能指示细胞的相对数量;③表达荧光素酶的骨髓间充质干细胞在颅骨缺损区局限分布,其生物荧光强度逐渐减弱,最终在术后35 d全部消失;④结果说明,生物发光成像是一种高效灵敏的活体示踪技术,有利于表征移植干细胞在体内的分布和数量变化,并为解释干细胞功能和机制研究提供有力证据。BACKGROUND:Bioluminescence imaging is an emerging in vivo imaging technology,but there are few studies on the tracing of stem cells after transplantation into living animals in existing reports.OBJECTIVE:To explore the multiplicity of infection of rat derived bone marrow mesenchymal stem cells transfected with Luciferase lentivirus,and further study the effectiveness and stability of Luciferase+bone marrow mesenchymal stem cells in vitro and the tracing effect in rat calvarial defects.METHODS:The logarithmic growth phase of rat bone marrow mesenchymal stem cells was transfected with different multiplicities of infection,and the fluorescence value was quantitatively analyzed in the in vivo imaging system to determine the optimal multiplicity of infection.Luciferase+bone marrow mesenchymal stem cells were transfected under optimal conditions,and fluorescence values corresponding to different numbers of cells and different passages were analyzed to assess the effectiveness and stability of Luciferase+bone marrow mesenchymal stem cells.Finally,Luciferase+bone marrow mesenchymal stem cells were traced in the rat calvarial defect model.RESULTS AND CONCLUSION:(1)When the multiplicity of infection increased in the interval of 10-50,the fluorescence quantification increased with the same proportion of multiplicity of infection.The fluorescence quantification did not increase significantly when the multiplicity of infection increased to 100,demonstrating that the optimal multiplicity of infection of Luciferase lentivirus transfected bone marrow mesenchymal stem cells was 50.(2)Luciferase+bone marrow mesenchymal stem cells had a good cell-bioluminescence dose-response relationship and proliferation stability in vitro,demonstrating that fluorescence quantification can indicate the relative number of cells.(3)The transplanted Luciferase+bone marrow mesenchymal stem cells were locally distributed in the skull defect,and the biofluorescence intensity decreased over time and finally disappeared after 35 days.(4)Bioluminescence i

关 键 词：干细胞 骨髓间充质干细胞 Luciferase慢病毒 生物发光成像 活体成像系统 活体示踪 大鼠 颅骨缺损 组织工程 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]